Molecular typing of HLA genes using whole genome amplified DNA.
BACKGROUND
The outcome of clinical transplantation and a number of disease susceptibilities show very strong associations with genetic variants within the major histocompatibility complex, particularly in the human leukocyte antigen (HLA) genes. A problem with many association studies is the lack of sufficient DNA to perform multiple genetic analyses, particularly with transplantation outcomes where donor and recipient DNA are often in short supply. This study assesses whether a multiple-strand displacement whole genome amplification (WGA) method could generate sufficient template of high quality to perform unbiased amplification for analysis of the HLA-A, -B, -C, -DRB1, and -DQB1 genes.


METHODS
A panel of DNA samples from various biological sources was subjected to WGA reaction using Phi29 DNA polymerase. The HLA genotypes were subsequently determined using standard polymerase chain reaction (PCR)-based methods including sequence-specific oligonucleotide probes (PCR-SSOP, Luminex, Luminex Corp.) and sequence-based typing (PCR-SBT). WGA products and original DNA samples were used to determine the sensitivity of the Luminex assay; in addition, reamplified WGA products were also genotyped.


RESULTS
The WGA templates, as well as serially amplified DNA for two successive rounds, yielded HLA genotypes fully concordant with those determined for the original DNA samples. WGA products and original DNA gave reproducible HLA-DQB1 genotypes with 100 to 10 ng of template. Purification of the WGA products was required for successful PCR-SBT, but not for the PCR-SSOP method.


CONCLUSIONS
Our study suggests that WGA can be a reliable method for generating unlimited DNA for medium- or high-resolution HLA typing using the techniques described above.
Creary
Lisa E
LE
Histocompatibility and Immunogenetics Research Group, Department of Histocompatibility and Immunogenetics, Colindale Centre, NHSBT, NHSBT, London, UK.
Girdlestone
John
J
Zamora
Jorge
J
Brown
Juliette
J
Navarrete
Cristina V
CV
D015894:Genome, Human; D005838:Genotype; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity